PROJECT SUMMARY Prostate cancer (PC) is the most common malignancy among males worldwide, and is the second leading cause of cancer death among men in United States. A man's life-time risk of developing PC is one in seven. Importantly, androgen receptor (AR) signaling is vital not only for the initiation of PC, which is initially androgen-dependent, but also for castration-resistant disease. Enzalutamide (ENZ) is the first and only FDA- approved second-generation AR antagonist used for the treatment of both non-metastatic and metastatic castration-resistant PC (CRPC). However, nearly all PC patients develop resistance to androgen-deprivation therapy (ADT). It has been well established that the progression of PC to castration-resistant growth and development of ADT resistance is associated with aberrant activation of Akt/mTOR signaling. Importantly, PI3K/Akt/mTOR axis acts as a transcriptional integrator of the androgen signaling pathway in PC cells. For many years, the cholesterol homeostasis in PC has been one of the main focuses of research. The consistent interest in the relationship between cholesterol and PC was largely driven by cholesterol involvement in de novo androgen synthesis. Our preliminary studies indicate that major cholesterol fractions (i.e. LDL, HDL, and VLDL) augment activation of Akt/mTOR signaling in PC cells, which coincides with resistance to ENZ. Sensitivity to ENZ was successfully reinstated following treatment with Akt/mTOR inhibitors. Based on these findings, we hypothesize that in addition to serving as a precursor for androgen biosynthesis, lipoprotein- derived cholesterol may promote castration-resistant growth of PC cells via androgen-independent activation of Akt/mTOR pathway. To test our hypothesis and to validate the therapeutic value of targeting cholesterol homeostasis in PC cells in order to increase the antitumor efficacy of ENZ, we propose the following Specific Aims: (1) To establish the role of cholesterol homeostasis in enzalutamide resistance; (2) To improve the antitumor efficacy of enzalutamide by targeting cellular cholesterol homeostasis. We anticipate that the proposed studies will gain insights that suggest new therapeutic options for the treatment of PC.